Dissection of membrane-binding and -remodeling regions in two classes of bacterial phospholipid N-methyltransferases

Abstract

Bacterial phospholipid N-methyltransferases (Pmts) catalyze the formation of phosphatidylcholine (PC) via successive N-methylation of phosphatidylethanolamine (PE). They are classified into Sinorhizobium-type and Rhodobacter-type enzymes. The Sinorhizobium-type PmtA protein from the plant pathogen Agrobacterium tumefaciens is recruited to anionic lipids in the cytoplasmic membrane via two amphipathic helices called αA and αF. Besides its enzymatic activity, PmtA is able to remodel membranes mediated by the αA domain. According to the Heliquest program, αA- and αF-like amphipathic helices are also present in other Sinorhizobium- and Rhodobacter-type Pmt enzymes suggesting a conserved architecture of α-helical membrane-binding regions in these methyltransferases. As representatives of the two Pmt families, we investigated the membrane binding and remodeling capacity of Bradyrhizobium japonicum PmtA (Sinorhizobium-type) and PmtX1 (Rhodobacter-type), which act cooperatively to produce PC in consecutive methylation steps. We found that the αA regions in both enzymes bind anionic lipids similar to αA of A. tumefaciens PmtA. Membrane binding of PmtX1 αA is enhanced by its substrate monomethyl-PE indicating a substrate-controlled membrane association. The αA regions of all investigated enzymes remodel spherical liposomes into tubular filaments suggesting a conserved membrane-remodeling capacity of bacterial Pmts. Based on these results we propose that the molecular details of membrane-binding and remodeling are conserved among bacterial Pmts.

Protein Amphipathic Helix Insertion: A Mechanism to Induce Membrane Fission

“…Among 16 AHs shown in the Table 1 of Drin and Antonny (2010), this motif is present only in α-synuclein, a protein that binds cardiolipin (Nakamura et al, 2008;Robotta et al, 2014;Ryan et al, 2018). However, except proteins listed in the Table 3, this motif is present in the AHs of at least two proteins that interact with cardiolipin but, to the best of our knowledge, are not known to be involved in membrane fission: PmtA from Bradyrhizobium japonicum (Danne et al, 2017a) and ATG3 (Nath et al, 2014;Hervás et al, 2017). Arg14 and Phe21 belong to TABLE 3 | Amphipathic helices of fission-inducing proteins that use cardiolipin as a lipid cofactor.…”

“…Amphipathic helix References the (K/R)x 6 (F/Y) motif in the AH of PmtA from B. japonicum (Danne et al, 2017a), whereas Lys11 and Tyr18 belong to the (K/R)x 6 (F/Y) motif in the AH of Atg3 (Hervás et al, 2017). In the AH of cardiolipin-binding fission-inducing protein PmtA from A. tumefaciens (Danne et al, 2017b), both Lys12 and Phe19 belonging to the Kx 6 F motif are critical for membrane binding (Danne et al, 2015).…”

A bacterial membrane sculpting protein with BAR domain-like activity

“…This shows that the BdpA homologs identified by PSI-BLAST form a cohesive group with most sequences forming clades by genus. The four unique BAR domain sequences from the five BdpA homologs predicted to contain a BAR domain based on the current model were subsequently aligned with representative known BAR domain-containing proteins from the various BAR domain subtypes (N-BAR, F-BAR, and I-BAR) ( Salzer et al, 2017 ), bacterial proteins with known membrane remodeling phenotypes ( Tanaka et al, 2010 ; Danne et al, 2017a ; Löwe and Amos, 1998 ), along with the most diverse ( Lu et al, 2020 ) BAR domain CDD sequences from eukaryotes ( Supplementary file 2 ). In this maximum likelihood phylogenetic tree generated from this alignment, the BdpA sequences form a cohesive clade separate from other bacterial proteins that associate with membranes or influence curvature in vitro and instead clusters more closely with eukaryotic BAR domain-containing proteins ( Figure 5 ).…”

“…Bacterial cell membrane curvature is observed during the formation of OMVs and OMEs, and it is proposed that proteins are necessary to stabilize these structures ( Bohuszewicz et al, 2016 ). Several bacterial proteins have demonstrated membrane tubule formation capabilities in vitro ( Tanaka et al, 2010 ; Danne et al, 2017a ; Danne et al, 2017b ; Danne et al, 2015 ; Low et al, 2009 ; Low and Löwe, 2006 ), but despite the growing number of reports, proteins involved in shaping bacterial membranes into OMV/OMEs in living cells have yet to be identified.…”

“…Despite our knowledge of numerous eukaryotic BAR domain-containing proteins spanning a variety of modes of curvature formation, membrane localizations, and subtypes, characterization of a functional bacterial BAR domain protein has yet to be reported. Previously described membrane curvature-promoting proteins in bacteria are unrelated to BAR domain proteins or contain features ancillary to eukaryotic BAR domain protein function, such as amphipathic alpha helices (SpoVM, PmtA) ( Danne et al, 2017a ; Danne et al, 2017b ; Gill et al, 2015 ; Kim et al, 2017 ), GTPase-associated signaling (FtsZ) ( Löwe and Amos, 1998 ), or positive curvature localization (SpoVM, MamY) ( Gill et al, 2015 ; Kim et al, 2017 ; Toro-Nahuelpan et al, 2019 ). However, actin-like ( van den Ent and Löwe, 2000 ; Pichoff and Lutkenhaus, 2005 ), dynamin-like ( Low and Löwe, 2006 ), and ESCRT-II-like ( Junglas, 2021 ; Liu, 2020 ) proteins have been previously discovered in bacteria and archaea, suggesting that vesicle formation and tubule biogenesis can be achieved through evolutionarily conserved mechanisms.…”